Figure 2

NSC114792 selectively inhibits JAK3 kinase activity in vitro. JAK1 (A), JAK2 (B), JAK3 (C), or TYK2 (D) immunoprecipitates were used for in vitro kinase assay. Each immunoprecipitate was pre-incubated with either vehicle (DMSO) alone, NSC114792 at different concentrations or the pan-JAK kinase inhibitor AG490 (100 μmol/L) for 1 hour. Kinase reactions were subsequently performed by the addition of recombinant STAT3α protein as a substrate and 2 μmol/L ATP for 30 minutes at 30°C. The reaction products were processed for Western blotting and probed with antibodies specific for phospho-STAT3, STAT3, JAK1, JAK2, JAK3, and TYK2. Note that NSC114792 blocks JAK3 kinase activity in a dose-dependent manner but failed to inhibit the kinase activity of other JAK family members. STAT3, JAK1, JAK2, JAK3, and TYK2 serve as a loading control.